Reduction of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase by Antisense RNA in the C4 Plant Flaveria bidentis Leads to Reduced Assimilation Rates and Increased Carbon Isotope Discrimination

Abstract

Transgenic Haveria bidentis (a C, species) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were used to examine the relationship between the CO, assimilation rate, Rubisco content, and carbon isotope discrimination. Reduction in the amount of Rubisco in the transgenic plants resulted in reduced CO, assimilation rates and increased carbon isotope discrimination of leaf dry matter. The H,O exchange was similar in transgenic and wild-type plants, resulting in higher ratios of intercellular to ambient CO, partial pressures. Carbon isotope discrimination was measured concurrently with C O , and H,O exchange on leaves of the control plants and T, progeny with a 40% reduction in Rubisco. From the theory of carbon isotope discrimination in the C, species, we conclude that the reduction in the Rubisco content in the transgenic plants has led to an increase in bundle-sheath CO, concentration and CO, leakage from the bundle sheath; however, some down-regulation of the C, cycle also occurred.The C, photosynthetic pathway requires the coordinated functioning of mesophyll and bundle-sheath cells within a leaf. Reactions of the C, cycle concentrate CO, in the bundle-sheath cells for assimilation by Rubisco (Hatch, 1987). This enhances Rubisco carboxylation while at the same time inhibiting Rubisco oxygenation, thus reducing the amount of photorespiration. The energy cost of this C0,-concentrating mechanism is 2 mo1 of ATP per regeneration of 1 mo1 of the primary CO, acceptor PEP, and any CO, that leaks from the bundle sheath rather than being fixed by Rubisco reduces the efficiency of the concentrating mechanism (Hatch, 1987). The compartmentation of the photosynthetic process between mesophyll and bundlesheath cells has complicated the study of C, photosynthesis, and the biophysical characterization of the C0,-concentrating function of C, photosynthesis has been experimentally difficult. Many of the parameters, such as bundle-sheath CO, concentration or the leakiness (4) of the bundle sheath (defined as the fraction of CO, generated by

Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

“…This effect on CO 2 assimilation rate is similar to that observed in F. bidentis plants with reduced Rubisco small subunit levels (Furbank et al, 1996;von Caemmerer et al, 1997). As predicted in the C 4 model, the CO 2 -saturated portion of an A/Ci curve (response of CO 2 assimilation rate to intercellular CO 2 ) in C 4 plants at high irradiance is naturally limited by either PEP or ribulose 1,5-bisphosphate regeneration or by maximum Rubisco activity (Berry and Farquhar, 1978;von Caemmerer and Furbank, 1999).…”

“…The similarity of maximum CO 2 assimilation rates in wild-type and antisense plants exhibiting greater than 30 mmol CO 2 m 22 s 21 NADP-ME activity suggests that in wild-type leaves the C 4 NADP-ME is either present in excess or is regulated to limit its activation, as observed with other C 4 enzymes. In a-SSu lines, both Rubisco content and maximal activity were shown to correlate linearly with CO 2 assimilation rates under saturating illumination (Furbank et al, 1996;von Caemmerer et al, 1997), indicating that Rubisco was a major limitation for maximal photosynthetic flux, with a control coefficient of 0.5 to 0.7. In contrast, extractable activities of NADP-malate dehydrogenase have been shown to be 10 times that required to support CO 2 assimilation rates (i.e.…”

What Does It Take to Be C4? Lessons from the Evolution of C4 Photosynthesis

“…Anti-sense experiments with C 4 Flaveria show that in spite of the CO 2 concentrating mechanism, Rubisco remains the major determinant of carbon flux at high light and moderate temperature in C 4 plants (18), with PEPCase and pyruvate-orthophosphate dikinase showing lower control coefficients. The complex regulatory cascades of many C 4 enzymes may be exercised more commonly as light-dark switches than as flux control systems during photosynthetic CO 2 fixation.…”

Bundle Sheath Leakiness and Light Limitation during C4 Leaf and Canopy CO2 Uptake

“…Alternatively, Henderson et al (1992) stated that with reduced phosphoenolpyruvate regeneration slowing C 4 cycle activity under low light, a decrease in the BS CO 2 concentration or CO 2 -O 2 ratio would allow the relative contribution of photorespiration to increase. The extra energy needed for the photorespiratory cycle would limit C 3 cycle activity compared with C 4 cycle activity, which would lead to an increase in leakage of CO 2 (Von Caemmerer et al, 1997a, 1997b) from BS cells, thereby allowing more of the isotopic fractionation by Rubisco (30&) to be expressed. Recently, Tazoe et al (2008) may have provided evidence for an increase in f as a result of energy limitation under low PFD, since the increase in D…”